MglB Is a Guanine Nucleotide Hydrolysis Activating Protein (GAP) for MglA

In eukaryotic cells, small G-proteins are critically regulated by Guanine nucleotide Exchange Factors (GEFs) and GTPase Activating Proteins (GAPs) [20],[21]. We have previously established that MglA can hydrolyze GTP, albeit at very slow rates in vitro, suggesting that an MglA-regulator with GAP activity could exist [18]. Based on sequence conservation, extensive search of the Myxococcus genome did not reveal eukaryotic-like regulators. Yet MglA is co-expressed with MglB, the founder member of a family of proteins that contains a so-called roadblock domain [22],[23]. The function of roadblock domains has not been demonstrated experimentally, but bioinformatics suggested that they might regulate the activity of a cognate NTPase [23]. Thus, MglB and MglB-like proteins could be prokaryotic regulators of bacterial small G-proteins such as MglA. To test this possibility, we purified recombinant MglA and MglB proteins to analyze in vitro whether MglB could act as a GAP for MglA (Figure 1A). In vitro, MglA bound but did not significantly hydrolyze radio-labeled GTP (Figure 1B and 1C). This result is consistent with previous results, showing that MglA hydrolyzed GTP at an extremely low rate in an enzyme-coupled assay [18]. In marked contrast, addition of MglB stimulated GTP hydrolysis by MglA in a dose-dependent manner (Figure 1C). MglB alone did not bind radioactive GTP (Figure 1B). Additionally, MglB was not found to affect GDP/GTP exchange on MglA: MglB stabilized the GTP bound form slightly (like a classical G-protein effector, Figure 1B) but did not modify the GDP off rate of MglA (unpublished data). Thus, MglB is an MglA GAP, which functions by switching MglA-GTP to MglA-GDP.

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Figure 1. MglB is a guanine nucleotide hydrolysis activating protein for MglA.

(A) Coomassie-stained gel showing purified MglA and MglB used in the biochemical assays. (B) MglB does not bind to non-hydrolysable GTPγS but stabilizes binding of MglA to GTPγS. Purified MglA (1 µM) and MglB (1 µM) were tested for their ability to bind GTPγS as described in experimental procedures. The presence of MglB does not accelerate the binding of GTPγS to MglA but increases the total amount of MglA bound to GTPγS by a factor of ∼2-fold. (C) Dose dependent activation of the MglA GTPase activity by MglB. Hydrolysis of GTP was measured by measuring ³²Pi release as described in experimental procedures. (D) The Q82L mutation renders MglA insensitive to the GAP activity of MglB. GTP hydrolysis by MglA and MglA_Q82L following MglB addition was measured over time as in (C).

https://doi.org/10.1371/journal.pbio.1000430.g001

MglB Is an Inhibitor of Cellular Reversals

What is the function of MglB in vivo? mglAB are encoded within a putative operon. In a previous work, deletion of mglB resulted in a dramatic reduction of the MglA levels, which precluded in-depth study of the function of MglB [24]. MglB was proposed to have a chaperone function for MglA explaining the observed lack of MglA stability [24], yet the low levels of MglA expression could also have been due to polar effects of the mglB deletion. To test the function of mglB in vivo, we deleted the region encoding residues 10–159 of mglB (MglB contains 159 residues). To show that this mglB deletion created no downstream polar effects on the expression of mglA, we successfully complemented the ΔmglB mutant by integrating another copy of mglB at an ectopic site on the Myxococcus chromosome (the Mx8-phage attachment site, Figure 2A and Figure S1A). Likewise, deletion of mglA was fully complemented when mglA was expressed from the Mx8-phage attachment site (Figure S1B). Western blots using anti-MglA and anti-MglB antibodies showed that MglA and MglB were produced stably in the ΔmglB and ΔmglA mutant, respectively (Figure 2A). We conclude that MglA and MglB are stable independently from each other and that their respective functions can be studied with the deletion mutants.

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Figure 2. MglA is the most downstream component of a pathway involving Frz and MglB.

(A) Levels of MglA and MglB expression in WT, ΔmglA (ΔA), ΔmglB (ΔB), and the respective complemented (ΔA+A, ΔB+B) strains assayed by Western blotting with anti-MglA and anti-MglB antibodies. (B) Motility phenotypes of WT, ΔmglB, ΔmglA, and ΔmglAB strains. Colony edges after 48 h incubation on hard (1.5%) agar are shown. Insets: colonies on soft (0.5%) agar, a substratum that only allows S-motility, visible at the edges. Scale bar = 1 mm. (C) Box plots of measured reversal frequencies in the various strains. The orange bar represents the average reversal frequency of each population. R: number of reversals scored in 30 min. E: ΔfrzE, Z: ΔfrzZ, BE: ΔmglB ΔfrzE, BZ: ΔmglB ΔfrzZ, CB: frzCD^c ΔmglB, B: ΔmglB, Q: mglA_Q82L, BQ: ΔmglB mglA_Q82L, EBQ: ΔfrzE ΔmglB mglA_Q82L, EQ: ΔfrzE mglA_Q82L, C: frzCD^c. n, number of cells that were tracked. (D) Traveled distances between reversals in different strains. d, ratio of the traveled distance over the cell length. A ratio of 1 indicates that the cells reverse after moving a distance corresponding to one cell length. Strains are labeled as in (C). n = 10 for each strain.

https://doi.org/10.1371/journal.pbio.1000430.g002

We tested the motility of the ΔmglB, ΔmglA, and ΔmglAB mutants in the hard (testing both A- and S-motility) and soft (testing S-motility only) agar assays. Swarming of the ΔmglB mutant was severely defective but detectable on both substrata (Figure 2B). The ΔmglA and ΔmglAB mutants looked completely non-motile under all conditions, showing that mglA acts downstream from mglB (Figure 2B). The motility defect of the ΔmglB mutant may be due to defects of the motility engines, aberrant directional control, or both. Time-lapse analysis of ΔmglB motile cells revealed that the cells moved with WT velocities (unpublished data). Strikingly, the ΔmglB mutant displayed an altered reversal frequency and reversed their direction of movement more frequently than WT cells (Figure 2C). Thus, MglB acts upstream from MglA and inhibits cellular reversals.

MglB Acts Downstream from the Frz-Signal Transduction Pathway

The Frz pathway regulates the reversal frequency of Myxococcus cells [25]. We wondered where MglB acts in the pathway. We combined frzE-null (kinase null) and frzZ-null (response-regulator null) mutations with a ΔmglB deletion and scored the reversal frequency of the double mutants. Strikingly, the ΔmglB mutation restored reversals of both the ΔfrzZ and ΔfrzE mutants (Figure 2C). The reversal frequency of the double mutants was significantly higher than the reversal frequency of WT cells yet remained slightly lower than the reversal frequency of the ΔmglB mutant (Figure 2C). To confirm the epistastic relationship between mglB and frz, we also combined a frzCD^c mutation (a mutation that hyperactivates Frz signaling) with the ΔmglB deletion. Both mutants hyper-reverse but they have significantly distinct reversal frequencies: the ΔmglB mutant has an average frequency of ∼10 rev.hour⁻¹, while the frzCD^c has an average frequency of ∼40 rev.hour⁻¹ (Figure 2C). A frzCD^c ΔmglB mutant reversed with frequencies that were indistinguishable from the ΔmglB mutant (∼10 rev.hour⁻¹), confirming that mglB acts downstream from the Frz-pathway. Agar swarming assays showed that the swarming pattern of the ΔfrzE ΔmglB was almost identical to the swarming pattern of the ΔmglB mutant, confirming that mglB acts downstream from the Frz pathway (Figure S2).

To clarify whether the ΔmglB mutant is indeed distinct from the ΔfrzE ΔmglB mutant, we measured the average distances traveled by the cells between reversals (Figure 2D). We found that the ΔfrzE ΔmglB mutant cells moved on average a distance corresponding to 4–5 cell lengths before they reversed (versus ∼8 cell lengths for the WT, Figure 2D). On the contrary, the ΔmglB mutant cells almost systematically reversed after moving a distance corresponding to one cell length (Figure 2D, see below).

Taken together, these results suggest that the Frz pathway activates cellular reversals by relieving an inhibition that mglB exerts on mglA: low reversal frequencies in frz-null mutants can thus be explained by a failure to relieve MglB inhibition, a mechanism that depends on FrzZ. However, Frz must also be able to signal to MglA independently from MglB because Frz-dependent regulation (albeit highly abnormal) is still detected when the reversal frequency of ΔmglB mutant is compared to the reversal frequency of double ΔfrzE ΔmglB mutant (see Discussion). This branching in the signaling pathway must occur downstream from FrzZ because ΔfrzZ ΔmglB mutants reverse with frequencies that are similar to the ΔfrzE ΔmglB (Figure 2C).

MglB Localizes at the Lagging Cell Pole and Oscillates in a Frz-Dependent Manner

To further understand the role of MglB, we generated a functional MglB-YFP fusion to investigate MglB dynamics during the reversal cycle (Figure S1). A single MglB-YFP focus was observed at the lagging cell pole and this focus switched systematically to the new lagging pole when cells reversed (Figures 3A, 3B, and S4A). Automated cross-correlation analysis confirmed that MglB oscillations are indeed coupled to cellular reversals (Figure 3C). Introduction of the frzCD^c allele led to hyper-oscillations of MglB-YFP (Figure 3D and 3E) whereas a frzE-null mutation abolished oscillations (unpublished data), confirming that MglB is indeed regulated by Frz.

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Figure 3. MglB-YFP localizes at the lagging cell pole.

(A) MglB-YFP switches upon cellular reversals. Scale bar = 2 µm. (B) Quantification of the relative fluorescence at the poles for the cell shown in (A) over time (min). Green line: initial leading pole. Orange line: initial lagging pole. R: reversal. (C) cross-correlation between MglB-YFP oscillations and cell reversals. (D) MglB-YFP dynamics are regulated by Frz. Oscillations of MglB-YFP in a frzCD^c mutant. Scale bar = 2 µm. (E) Quantification of the relative fluorescence at the poles for the cell shown in (D) over time (min). Green line: initial leading pole. Orange line: initial lagging pole. R: reversal.

https://doi.org/10.1371/journal.pbio.1000430.g003

MglA and MglB Oscillate Synchronously and Inversely

Our genetic and biochemical evidence suggests that MglB inhibits reversals by activating GTP hydrolysis by MglA at the lagging cell pole. In a previous work, we constructed a partially functional MglA-YFP chimera: cells expressing MglA-YFP alone were motile but significantly impaired in their reversal frequency, precluding studies of MglA dynamics during the reversal cycle (Figure S3 [18]). To monitor the dynamics of MglA-YFP in reversing cell, we expressed MglA-YFP in the presence of MglA. In these merodiploid cells, expression of MglA-YFP was not associated with detectable motility defects (see Protocol S1 for details on the construction and Figure S3). Thus, we conclude that MglA-YFP dynamics during reversal can be studied using the merodiploid system, which will be systematically used for the rest of this study.

In a fluorescent time-lapse motility assay, MglA-YFP localized at the leading cell and within fixed internal clusters (Figures 4A and S4B). MglA-YFP oscillated from pole to pole and was systematically redirected to the new leading pole at the time of reversal (Figure 4A and 4B). Cross-correlation analysis also confirmed that MglAYFP oscillations are coupled with the reversal cycle (Figure 4C). Thus, the localization of MglA-YFP is coupled to the reversal cycle. MglA-YFP dynamics were clearly regulated by the Frz pathway: in a ΔfrzE mutant oscillations of MglA-YFP were abolished (unpublished data), while they were enhanced and correlated with increased cellular reversals in a frzCD^c mutant (Figures 4D and S5), confirming that MglA is a downstream component of the reversal switching machinery.

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Figure 4. MglA and MglB oscillate inversely and synchronously.

(A) MglA-YFP switches to the new leading pole when cells reverse. Fluorescence and corresponding phase contrast overlays are shown. Triangles show MglA internal clusters. Scale bar = 2 µm. (B) Quantification of the relative fluorescence (grey values, arbitrary units) at the poles for the cell shown in (A) over time (min). Orange line: initial leading pole. Green line: initial lagging pole. R: reversal. (C) Cross-correlation between MglA-YFP oscillations and cell reversals. (D) Dynamics of MglA-YFP in the frzCD^c mutant. Legend reads as in (B). (E) Dynamics of MglA-YFP and MglB-mCherry in a reversing cell. Upper panel: MglA-YFP; middle panel: MglB-mCherry; lower panel: merge. Scale bar = 2 µm. (F) Quantification of the relative YFP (green) and mCherry fluorescence (red) at the initial leading pole for the cell shown in (E) over time (min).

https://doi.org/10.1371/journal.pbio.1000430.g004

To monitor the dynamic behaviors of MglA and MglB simultaneously, we engineered merodiploid cells expressing both MglA-YFP and a functional MglB-mCherry. As expected from analysis of the individual fusions in WT and frz backgrounds, both proteins oscillated inversely and synchronously, switching to opposite poles when cells reversed (Figure 4F and 4E).

MglB Is a Critical Determinant of Asymmetrical MglA-YFP Localization

We then analyzed the dynamic localization of MglA-YFP in absence of MglB. In this mutant, MglA-YFP did not localize to one cell pole but localized to both cell poles: minor fluctuations in MglA-YFP fluorescence were observed over time, but these changes were rapid and transient and not obviously correlated with the timing of reversals (Figure 5A and 5B). Thus, a function of MglB is to prevent MglA-YFP accumulation at the lagging pole, which seems to result in aberrant reversals.

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Figure 5. MglB prevents accumulation of MglA at the lagging cell pole.

(A) MglA-YFP is bipolar in absence of mglB. Fluorescence and corresponding phase contrast overlays of a ΔmglB mutant expressing MglA-YFP are shown. Scale bar = 2 µm. (B) Cross-correlation between MglA-YFP dynamics and cell reversals in the ΔmglB mutant (orange curve) and the WT strain (gray curve). (C) MglA_Q82L-YFP is bipolar. Fluorescence and corresponding phase contrast overlays of an mglA_Q82L mutant expressing MglA_Q82L-YFP are shown. Scale bar = 2 µm. (D) Cross-correlation between cell reversal and MglA_Q82L-YFP dynamics (orange curve) or MglA-YFP (gray curve).

https://doi.org/10.1371/journal.pbio.1000430.g005

MglB Regulates the Nucleotide State of MglA In Vivo

MglB could inhibit cellular reversals by catalyzing the transition from MglA-GTP to MglA-GDP at the lagging cell pole. The in vitro results show that MglA does not hydrolyze GTP significantly in absence of MglB; thus, MglA may be mostly GTP-bound in the mglB mutant. If so, a mutation that locks MglA in its GTP-bound form should mimic the mglB mutation. We designed an MglA mutant where Glutamine 82 is replaced by a Leucine, a mutation predicted to lock MglA in its GTP-bound state by inhibiting GTP hydrolysis (Figure S6A, [26]). In vitro, MglA_Q82L bound GTP stably like the WT MglA (Figure S6B), but contrarily to WT MglA and as expected, addition of MglB failed to activate hydrolysis, showing that MglA_Q82L cannot hydrolyze GTP (Figure 1D). When expressed in vivo, MglA_Q82L was found to be as stable as WT MglA (Figure S7).

If MglB prevents MglA binding at the lagging pole through its GAP activity, MglA_Q82L should localize at both cell poles despite the presence of MglB. As expected, an MglA_Q82L-YFP chimera (see Protocol S1 for details on this construction) was mostly found at both cell poles and the cells reversed in the absence of MglA_Q82L-YFP oscillations (Figure 5C and 5D), similarly to MglA-YFP in the ΔmglB mutant (Figure 5A and 5B). We conclude that the function of MglB is to catalyze the transition from MglA-GTP to MglA-GDP, preventing accumulation of MglA at the lagging cell pole and thus inhibiting reversal frequency.

The MglA GTP Cycle Determines Reversal Frequency

MglA_Q82L expressing cells reversed with a reversal frequency that was almost identical to that of the mglB deletion mutant (Figure 2C and 2D). In fact, similarly to the ΔmglB mutant, mglA_Q82L cells almost systematically moved exactly the length of one cell body before they reversed (Figure 2D). To confirm that reversals are regulated through the MglA GTP hydrolysis cycle, we measured the reversal frequencies of mglA_Q82L ΔmglB and mglA_Q82L ΔmglB ΔfrzE mutant cells. All mutants reversed with frequencies similar to the mglA_Q82L (Figure 2C) showing that the MglA GTP switch is the final downstream output of the Frz MglB transduction pathway.

The MglAB Module Creates Dynamic Cell Polarity

Our results suggest that the MglA and MglB proteins define the polarity switch that controls cellular reversals. However, an outstanding question remains: Why are cells still reversing in absence of a GTP hydrolysis cycle (for example in ΔmglB and mglA_Q82L cells)? This seems paradoxical because a simple assumption was that in absence of dynamic polarization, the cells would be non-reversing rather than hyper-reversing. A clue, however, is that in all cases the cells reverse after they moved the distance of one cell length (Figure 2D).

Since single motile cells move essentially by A-motility, we analyzed AglZ-YFP dynamics in the mglA_Q82L mutant. In WT cells, AglZ-YFP localizes at the leading pole and assembles within fixed adhesion clusters dispersed at the back of the cell (Figure S8 and [13]). In the mglA_Q82L mutant, AglZ-YFP was not significantly retained at the cell pole; instead, a major fluorescent cluster remained at a fixed position relative to the substratum at all times (Figure 6A). Cells systematically reversed once the AglZ-YFP clusters accumulated at the lagging cell end (Figure 6A). Comparable results were obtained in the mglB mutant (unpublished data). Thus, AglZ-YFP no longer oscillates between poles in absence of the MglA GTP-cycle. Instead, we propose that the cells reverse because the A-motility system is intrinsically capable to switch its own directionality, for example, once key regulatory proteins such as AglZ accumulate at the lagging cell pole (see Discussion).

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Figure 6. MglAB establish dynamic cell polarity.

(A) AglZ-YFP dynamics in an mglA_Q82L mutant. Triangles point to a major AglZ-YFP cluster that remains fixed at all times. Scale bar = 2 µm. (B) FrzS-YFP dynamics in an mglA_Q82L mutant. Scale bar = 2 µm. (C) Quantification of the relative fluorescence at the poles for the cell shown in (B) over time (min). Orange line: initial leading pole. Green line: initial lagging pole.

https://doi.org/10.1371/journal.pbio.1000430.g006

To confirm that the MglA GTP cycle is essential for dynamic cell polarity we also tracked the localization of the downstream S-motility protein FrzS-GFP in the mglA_Q82L strain. Under our experimental conditions, single cells do not move by S-motility but S-motility proteins dynamics are still coupled to the directionality of A-motile cells [11],[12]. For example, FrzS-GFP clusters at the leading cell pole and oscillates from pole to pole during the reversal cycle in wild type cells ([12] and Figure S9A). In both the mglA_Q82L and ΔmglB strains, FrzS-GFP localized to both cell poles but showed no detectable switching: fluorescence intensities fluctuated at the cell poles but these fluctuations did not correlate with the direction of movement (Figure 6B, 6C and Figure S9B). Finally, we also looked at the dynamics of the RomR-GFP protein, which belongs to the A-motility system and binds at the lagging end, oscillating inversely and synchronously with FrzS and AglZ [14]. In absence of MglB, RomR-GFP was bipolar and showed no oscillation, confirming the lack of dynamic cell polarity (Figure S10). All together, these results show that MglA and MglB establish a polarity axis that drives programmed cellular reversals.

How Does MglAB Create Dynamic Cell Polarity?

MglA-binding cues may in fact exist at both cell poles because MglA-GTP is bi-polar in absence of MglB regulation (i.e., MglA_Q82L or MglA in the mglB mutant). However, these cues are not dynamically regulated because bi-polar MglA does not oscillate with the reversal cycle. Also, FrzS and RomR localize non-dynamically to one cell pole in absence of MglA [14],[18]. Thus, targeting of motility proteins to the cell poles is probably wired into the cell cycle itself in a process similar to flagellar assembly at new division septa [30],[31]. Conceptually, polar curvature itself may play a role in recognition because small G-proteins and their regulators can bind curved membranes [32] and several proteins have been shown to recognize curvature at the bacterial poles [33],[34].

Several lines of evidence suggest that MglAB is the polarity generator that creates cellular reversals: (i) MglA acts downstream from the Frz pathway and switches systematically to the new leading pole. (ii) Expression of an MglA variant locked in its GTP-bound state is epistatic over Δfrz and ΔmglB mutations, showing that MglA is the most downstream component of the regulatory cascade that controls reversals. (iii) MglA interacts directly with FrzS and AglZ [18],[19] and is essential for the dynamic localization of FrzS, AglZ, and RomR. (iv) The perturbations of the MglA GTP cycle affect dynamic polarity of MglA itself and creates aberrant dynamic behaviors of the downstream proteins FrzS, AglZ, and RomR. (v) MglA is a bona fide small G-protein, a class of essential polarity regulators in eukaryotic cells. The results suggest that MglB acts to sequester MglA-GTP at the leading cell pole where it would activate both motility systems, for example, by engaging FrzS and AglZ (Figure 7A). Consistent with this, MglA_Q82L-YFP, a GTP-locked mutant, accumulates at the lagging cell pole, despite the presence of MglB. MglB may also trigger dispersal of the focal adhesion clusters by inactivating cluster-associated MglA and preventing uncontrolled A-motility directional switches (see below). Thus, a Frz-dependent mechanism could simply invert the polarity axis by switching MglB to the opposite cell pole (Figure 7A).

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Figure 7. Working model for the regulation of dynamic cell polarity in Myxococcus.

(A) Dynamic polarity switched during cellular reversals. See text for details. The orange yellow gradient symbolizes proposed MglA-GTP (orange) and MglA-GDP (yellow) distribution in the cell. At the time of reversal, an Frz-dependent unknown mechanism triggers MglA and MglB to switch to opposite poles. During the pause, the cell is depolarized, FrzS is symmetrically distributed at both cell poles, and the AglZ clusters are dispersed throughout the cytosol (green). The cycle is then reinitiated and the cell moves in the opposite direction. The sizes of the protein symbols reflect relative amounts at specific subcellular sites. (B) Cellular reversals in absence of an MglA GTP cycle. See text for details.

https://doi.org/10.1371/journal.pbio.1000430.g007

This model is attractive but still has a number of unresolved questions. How are MglA and MglB switched to generate reversals? MglA- and MglB-YFP do not accumulate gradually at opposite poles but rather are rapidly relocated at the time of reversal (within ∼30 s) (Figures 3B and 4B), arguing that a transient signal input triggers the switch. Interestingly, bursts of phosphorylated FrzZ are predicted by mathematical modeling [17]. If true, FrzZ may trigger re-localization of MglB to the opposite cell pole directly, potentially by inhibiting the MglB GAP activity. However, this scenario is probably over-simplistic: our results clearly point to the existence of additional MglA regulators: the “pendulum” motility of the ΔmglB mutant can be suppressed partially by deletion of either frzE or frzZ (Figure 2D), suggesting the existence of an additional Frz-modulated regulator of MglA. frz suppression does not occur in cells expressing the MglA GTP-locked variant; thus, the suppression mechanism specifically occurs at the level of the MglA GTP cycle (Figure 2C). In addition, MglA may be mostly in its GTP-bound form in the ΔmglB mutant because the reversal frequency and localization of MglA-YFP were very similar in the ΔmglB and mglA_Q82L mutants. Thus, frz suppression of the ΔmglB mutant pendulum motion argues that the MglA GTP-cycle is somewhat restored in the double mutant (albeit incompletely), for example because of an additional MglA GAP.

MglB Defines a Novel Family of Bacterial GAPs

Eukaryotic small G-proteins are often controlled through the balancing actions of GAPs and GEFs, each catalyzing opposite steps of the GTP switch [21]. A GEF may be necessary to switch MglA back to the GTP bound form after MglB activation of GTP hydrolysis, but MglA could also spontaneously switch back to the GTP-bound state if the GTP/GDP balance is favorable in the cell. Small GTPase regulation by bacterial proteins is common during pathogenesis where invasive bacteria inject effectors that mimic GAPs and GEFs directly into the host cell to disrupt small GTPase signaling [35]. However, none of these virulence factors are related to MglB and there is no evidence that they have a physiological function in bacteria that express them. Thus, MglB-like proteins are probably major regulators of bacterial small GTPases. How MglB activates GTP hydrolysis by MglA is an interesting question because MglB contains a widespread so-called LC7/roadblock domain [23]. LC7/roadblocks are ancient protein domains conserved in all three eukaryotic, prokaryotic, and archeal kingdoms, yet very little information is available about their function. It was suggested that the members of this family regulate NTPases because proteins that contain roadblock domains are almost invariably linked to an NTPase [23]. For example, the LC7 protein is a conserved component of the Dynein light chain and has an important regulatory role on the activity of this motor [36]. However, the lack of a simple biochemical system to test LC7 function has hampered our understanding of how these domains work. Thus, understanding how MglB regulates MglA at the molecular level is potentially of great significance to understand the function of a widely conserved protein domain.

The “Pendulum” Motion Highlights Unexpected Properties of the A-Motility Machinery

The oscillation dynamics of FrzS-GFP and RomR-GFP were abrogated in absence of the MglA-GTP cycle, suggesting that the MglAB proteins polarize the cell dynamically to regulate cellular reversals. Thus, we were surprised to find that the cells still reversed in absence of the MglA GTP-cycle. This was puzzling because the dynamics of RomR, an A-motility protein, are not coupled to the reversal cycle in the ΔmglB mutant. So how were these reversals generated if the cells are not dynamically polarized? Figure 7B depicts a proposed mechanism. In absence of the MglA GTP switch, active engine units may be assembled at the cell pole and produce movement. When these units reach the lagging cell end, they are not disassembled because MglB cannot activate MglA-GTP hydrolysis within the complexes. Instead, we propose that a threshold is reached at the pole, activating a built-in capacity in the machinery to reverse its directionality and resume movement in the opposite direction. The cycle can thus be repeated endlessly resulting in the “pendulum” behavior. The term “pendulum gliding” was borrowed from studies on Plasmodium sporozoites (apicomplexan parasites) [37]. We previously discussed that A-motility may be analogous to Apicomplexan gliding motility because the parasites assemble focal adhesion complexes at their apical end and glide forward by moving these adhesions rearward in an actomyosin motorized process [13],[38]. Strikingly, mutant sporozoites expressing a truncated version of the adhesion factor TRAP also move like a pendulum, most likely because they fail to relieve TRAP-dependent adhesions at their trailing end [37]. In Myxococcus, failure to relieve focal adhesions at the back of the cells could also trigger a switch to the opposite direction. Thus, a critical function of MglB is to suppress these periodic switches and allow the cell to move distances corresponding at least to several cell lengths before it reverses. The molecular basis for directional inversion needs investigation both in Plasmodium and Myxococcus.

In a previous work, we showed that A-motility complexes require the bacterial MreB actin cytoskeleton and MglA, much like focal adhesion complexes that drive eukaryotic cell migration [18]. Thus, the mechanisms underlying Myxococcus motility are remarkably similar to the mechanisms that drive eukaryotic cell motility. In Dictyostelium discoidum, chemotaxis involves a complex arsenal of receptor-activated small GTPases and their cognate regulators [1],[2]. In Myxococcus, polarity seems to be controlled by a single small G-protein switch that acts downstream of a chemotaxis-like signal transduction pathway. It is thus an ideal model system to dissect molecular regulations that may be extremely widespread. Finally, MglA and MglB homologues are also widespread in prokaryots [39], many of which are not motile, suggesting that small GTPase switches also regulate multiple biological processes in bacteria, a field of research that has not received the attention it deserves.

Bacterial Strains, Plasmids, and Growth

See Table S1 for plasmids and Table S2 for strains and their mode of construction. Primer sequences and plasmid construction schemes are provided in Tables S3 and S4. M. xanthus strains were grown at 32°C in CYE rich media as previously described [6]. Plasmids were introduced in M. xanthus by electroporation. Mutants and transformants were obtained by homologous recombination based on a previously reported method [6]. Complementation, expression of the fusion and mutant protein were all obtained after ectopic integration of the genes of interest at the Mx8-phage attachment site in appropriate deletion backgrounds (Table S2). For co-expression of both MglA_Q82L and MglA_Q82L-YFP, expression of MglA_Q82L was additionally driven from the car locus, another ectopic site with the pCT2 system (Table 1 in [40]). Both integration at Mx8_att and car have no effect on cell motility [14],[40].

For phenotypic assays, cells, at a concentration of 4×10⁹ cfu ml⁻¹, were spotted on CF-agar plates or CYE plates containing an agar concentration of 1.5%, incubated at 32°C, and photographed after 48 h with an Olympus SZ61 binocular or a Nikon Eclipse (model TE2000E) microscope.

Expression and Purification of MglA and MglB

MglA-His₆ and His₆-MglB were expressed from the expression vector pET28(a) (Novagen). Expression of the recombinant proteins was induced in both cases by growing cells at room temperature for 20 h in the presence of 0.5 mM IPTG (isopropyl-h-d-thiogalactopyranoside). Cells were then harvested by centrifugation at 8,000 rpm for 10 min, resuspended in a buffer containing 50 mM NaH₂PO₄ pH 8.0, 300 mM NaCl, 10 mM imidazol, and lysed with a French press. The lysates were centrifuged twice (18,000 rpm, 4°C, 30 min) to remove debris prior to the purification. Supernatants were incubated with Nickel beads for 1 h at 4°C and the beads were collected and loaded into 5 ml HisTrap™ nickel columns (GE Healthcare). The elution was performed by using a buffer containing 50 mM NaH₂PO₄ pH 8.0, 300 mM NaCl, 250 mM imidazol, and for MglA, GDP 30 µM. Eluates were finally dialysed against a storage solution containing 50 mM NaH₂PO₄ pH 8.0, 300 mM NaCl, 10% Glycerol, and for MglA, GDP 30 µM. Protein purity and stability was determined by a Bradford assay and SDS-PAGE. The recombinant proteins were used both to immunize rabbits and perform biochemical in vitro assays.

GTPγS-Binding Assay

Purified MglA, MglA_Q82L, and MglB (1 µM final) were incubated at 30°C with either 20 µM non-hydrolysable [³⁵S]GTPγS or 15 µM γ[³²P]GTP in 50 mM HEPES (pH 7.5), 100 mM KCl, 1 mM MgCl₂, 5 mM Pi, and 1 mM DTT. We found that, in solution, MglA is stabilized by addition of lipids; thus, all biochemical assays were conducted in the presence of 1 g/l Azolectin vesicles. Samples of 25 µl were filtered at the indicated times and radioactivity was counted [41]. The curves were obtained by fitting the data to the model y = A_o(1−e^−kt) with k = 0.29 min⁻¹ for MglA alone and k = 0.12 min⁻¹ for MglA in the presence of MglB.

GTPase Assay

Purified MglA and MglA_Q82L (1 µM final) were loaded with 15 mM [γ-³²P]GTP (∼1,400 cpm/pmoles) in 50 mM HEPES (pH 7.5), 100 mM KCl, 1 mM MgCl₂, 5 mM Pi, 1 mM DTT, in the presence of 1 g/l Azolectin (Sigma) vesicles for 4 min at 30°C. The GTP hydrolysis was initiated by the addition of 1 µM (unless otherwise stated) of purified MglB. At the indicated times, aliquots of 25 µl were removed. ³²Pi release was measured by the charcoal method [42]. Briefly, the 25 µl samples were added to 750 µl of 5%(w/v) charcoal (100–400 mesh, Sigma) in 50 mM NaH₂PO₄ (4°C) and vortexed. The charcoal was removed by centrifugation (5 min at 13.2 krpm) and the amount of radioactivity present in the supernatant was determined by liquid scintillation counting.

Western Blotting

Western blotting was performed as previously described [40] with 1/10,000 dilutions of MglA or MglB antisera.

Time Lapse Fluorescence Microscopy

Time lapse experiments were performed as previously described [43]. Microscopic analysis was performed using an automated and inverted epifluorescence microscope TE2000-E-PFS (Nikon, France). The microscope is equipped with “The Perfect Focus System” (PFS) that automatically maintains focus so that the point of interest within a specimen is always kept in sharp focus at all times, in spite of any mechanical or thermal perturbations. Images were recorded with a CoolSNAP HQ 2 (Roper Scientific, Roper Scientific SARL, France) and a 40×/0.75 DLL “Plan-Apochromat” or a 100×/1.4 DLL objective. All fluorescence images were acquired with a minimal exposure time to minimize bleaching and phototoxicity effects.

Cell tracking was performed automatically using a previously described macro under the METAMORPH software (Molecular devices); when appropriate, manual measurements were also performed to correct tracking errors with tools built into the software. Images were processed under both ImageJ 1.40g (National Institute of Health, USA) and METAMORPH.

Statistical Analysis

Cells (n>100) were automatically segmented by successive morphological operations: h-dome extraction, gray-scale reconstruction, binary images, and morphological opening. To optimize segmentation, binary frames were sometimes corrected manually with appropriate tools built into the software. A binary mask was then used to perform integrated morphometric analysis and cell tracking. Cell tracking was performed following standard mathematical procedures already described in [43]. Computational scoring of cell reversals was obtained by tracking cells that showed clear directional changes, moving at least a 10^th of their cellular length in the opposite direction. To correlate these reversals to changes in fluorescence at the cell poles, the cell poles were automatically detected using a custom automation script (Visual Basic) under Metamorph 7.5 (Molecular Devices, Molecular Devices France, France). In this system, polar fluorescence inversions were systematically scored when the difference between the average grey intensity values of the poles became significantly different from the standard deviation of the average intensity value along the length of the cell. All selected cells were verified manually to ensure that the automatic process always scored actual reversals and polar fluorescence inversions. The cross-correlation coefficient (Rxy) between scored reversals and fluorescence pole-to-pole switchings for a time of delay (m) was calculated with the following equation:The maximum value is Rxy = 1 for a perfect correlation. The time lapse movies are composed of 30 s time frames due to illumination constraints (toxicity and bleaching). In these conditions, a Rxy>0.5 for a time delay = 0 (±30 s) means that the two events, fluorescence polar inversions (x(t)) and cellular reversals (y(t)), are highly correlated.